Premature birth is the leading cause of perinatal mortality and morbidity worldwide. The Perinatology Research Branch has defined preterm labor as a syndrome and determined that at least 25% of all preterm births are born to women with sub-clinical intrauterine infection. Moreover, we have provided evidence that many premature neonates are critically ill before birth and proposed that the onset of premature labor has survival value in the context of intrauterine infection. The goal of this project is to understand the pathophysiology of premature labor and delivery and the focus of our research this year was to study the role of matrix degrading enzymes in preterm premature rupture of membranes (PROM). The fetal membranes normally rupture after the onset of spontaneous labor. PROM occurs in 10% of women at term and is associated with 30-40% of preterm deliveries. The mechanisms responsible for PROM are poorly understood. Histologic studies of the site of rupture have demonstrated a zone of altered morphology characterized by thickening of the connective tissue components of the membranes and thinning of the cytotrophoblast layer and decidua, as well as disruption of the interconnection between amnion and chorion. A significant decrease in the density of collagen type I, III and V has been reported in the zone of altered morphology, which is in close proximity to the cervix. The current hypothesis is that increased protease activity leads to an accelerated degradation of the extracellular matrix of the fetal membranes and that this, coupled with biomechanical forces associated with labor, eventually leads to membrane rupture. In the context of intrauterine infection, proteases produced by bacteria or host cells could decrease membrane strength and elasticity. This year the Branch made the following observations: 1. Neutrophil elastase, a multifunctional serine protease stored in azurophilic granules of mature neutrophils, capable of extracellular degradation of connective tissue during an inflammatory process, was determined in the amniotic fluid of women with and without rupture of membranes. We found that preterm PROM was associated with a significant increase in the amniotic fluid concentration of neutrophil elastase, as was microbial invasion of the amniotic cavity. In contrast, preterm PROM was not associated with changes in the concentrations of Stromelysin 1 [matrix metalloproteinase (MMP)-3], which was only elevated in the context of infection. These observations add to previous work of the Branch suggesting the participation of MMP-1, MMP-8, MMP-9, but not MMP-2, MMP-3, MMP-7, and MMP-13 in the mechanisms of membrane rupture. Collectively, there appears to be selective participation of proteases in the process of membrane rupture in vivo. 2. Functional polymorphisms for genes encoding for MMP-1 and MMP-9 have been recently recognized. Two studies were conducted to determine if these polymorphisms would increase the expression of MMP-1 and MMP-9 by amnion cells, and whether their frequency may be increased in patients with preterm PROM. A polymorphism at -1607 in the MMP-1 promoter [an insertion of a guanine (G)] creates a core Ets binding site and increases promoter activity. The 2G promoter had >2-fold activity than the 1G allele in amnion mesenchymal cells and a clone amnion cell line. Phorbol 12-myristate 13-acetate (PMA) increased mesenchymal cell nuclear protein binding with greater affinity to the 2G allele. Induction of MMP-1 mRNA by PMA was significantly greater in cells with a 1G/2G or 2G/2G genotype compared with cells homozygous for the 1G allele. When treated with PMA, the 1G/2G and 2G/2G cells produced greater amounts of MMP-1 protein than 1G/1G cells. A significant association was found between fetal carriage of a 2G allele and preterm PROM. Thus, the 2G allele has stronger promoter activity in amnion cells, it confers increased responsiveness of amnion cells to stimuli that induce MMP-1, and carriage of this polymorphism increases the risk of preterm PROM. The functional significance of a variable number tandem repeat and a single nucleotide polymorphism (SNP) in the MMP-9 gene on promoter activity and their association with preterm PROM were studied. The 14 CA-repeat allele was a stronger promoter than the 20 CA-repeat allele in amnion epithelial cells and WISH amnion-derived cells, but in THP-1 monocyte/macrophage cells the 14 and 20 CA-repeat alleles had similar activities. An SNP at -1562 did not significantly affect promoter activity. A case-control study indicated that the 14 CA-repeat allele was more common in newborns delivered to mothers who had preterm PROM than in those delivered at term. There was no association between the -1562 SNP and preterm PROM. Thus, the evidence suggests that there are cell host-dependent differences in MMP-9 promoter activity related to the CA-repeat number and that fetal carriage of the 14 CA-repeat allele is also associated with preterm PROM.